Fecal Bacteria Implicated in Biofilm Production Are Enriched and Associate to Gastrointestinal Symptoms in Patients With APECED - A Pilot Study

Abstract

Backgrounds and aims: APECED is a rare autoimmune disease caused by mutations in the Autoimmune Regulator gene. A significant proportion of patients also have gastrointestinal symptoms, including malabsorption, chronic diarrhea, and obstipation. The pathological background of the gastrointestinal symptoms remains incompletely understood and involves multiple factors, with autoimmunity being the most common underlying cause. Patients with APECED have increased immune responses against gut commensals. Our objective was to evaluate whether the intestinal microbiota composition, predicted functions or fungal abundance differ between Finnish patients with APECED and healthy controls, and whether these associate to the patients' clinical phenotype and gastrointestinal symptoms.

Methods: DNA was isolated from fecal samples from 15 patients with APECED (median age 46.4 years) together with 15 samples from body mass index matched healthy controls. DNA samples were subjected to analysis of the gut microbiota using 16S rRNA gene amplicon sequencing, imputed metagenomics using the PICRUSt2 algorithm, and quantitative PCR for fungi. Extensive correlations of the microbiota with patient characteristics were determined.

Results: Analysis of gut microbiota indicated that both alpha- and beta-diversity were altered in patients with APECED compared to healthy controls. The fraction of Faecalibacterium was reduced in patients with APECED while that of Atopobium spp. and several gram-negative genera previously implicated in biofilm formation, e.g. Veillonella, Prevotella, Megasphaera and Heamophilus, were increased in parallel to lipopolysaccharide (LPS) synthesis in imputed metagenomics. The differences in gut microbiota were linked to patient characteristics, especially the presence of anti-Saccharomyces cerevisiae antibodies (ASCA) and severity of gastrointestinal symptoms.

Conclusions: Gut microbiota of patients with APECED is altered and enriched with predominantly gram-negative bacterial taxa that may promote biofilm formation and lead to increased exposure to LPS in the patients. The most pronounced alterations in the microbiota were associated with more severe gastrointestinal symptoms.

Keywords: atopobium; autoantibodies; autoimmunity; dysbiosis; faecalibacterium; gut microbiota; immune dysregulation; lps.

Figure 1

(A) Principal coordinate analysis (PCoA) based on Bray–Curtis distances using the phylum-level data showing differences in the gut microbiota compositions between patients with APECED (black) and healthy controls (red). (B) The relative abundances of Firmicutes, Bacteroidetes and Proteobacteria in patients with APECED (open circles) compared to healthy controls (black circles). * indicates FDR-P-value < 0.05 (C) Composition of the gut microbiota at the family level of patients with APECED and healthy controls. For readability, the composition of the controls is shown as mean relative abundance. Anti-Saccharomyces cerevisiae antibodies (ASCA), antibodies against tryptophan hydroxylase (TPH), gastrointestinal symptoms and results of stool yeast analysis are shown for each patient. – negative/low, + positive, ++ markedly positive i.e. severe symptoms, ~ average.

Figure 2

The relative abundance of all the bacterial genera that significantly altered in APECED patients’ stool (open circles) compared to healthy controls (black circles) based on the age-adjusted negative binomial models. The results of statistical analysis are summarized in Table 2 .

Figure 3

(A) Principal coordinate analysis (PCoA) based on Bray–Curtis distances using the predicted functional modules showing differences in the gut microbiota compositions between patients with APECED (black) and healthy controls (red). (B) Predicted functional modules significantly overrepresented in patients with APECED (black) and controls (red). (C) Receiver operating characteristic (ROC) curves of the cross-validated random forest models constructed using the imputed functions (red) and relative abundances of genera (green).

Figure 4

(A) Principal coordinate analysis (PCoA) based on Bray–Curtis distances using the phylum-level data showing differences in the gut microbiota compositions between ASCA-positive and ASCA-negative patients with APECED. (B) The relative abundance of Firmicutes in patients with or without ASCA. (C)Principal coordinate analysis (PCoA) based on Bray–Curtis distances using the genus-level data showing differences in the gut microbiota compositions between patients with APECED ranked according to abundance of yeast in stool and the severity of patient’s gastrointestinal symptoms, respectively. *** denotes p-value < 0.001; –, negative/low; +, positive; ++, markedly positive ie. severe symptoms; ~, average.